This invention relates to antisense inhibition of translation of Hepatitis C Virus RNA.
Antisense oligonucleotides (oligos) are useful tools for studying cellular and viral gene function (Uhlmann et al., Chem. Rev. 90(4):543-584, 1990; Stein et al., Science 261:1004-1012, 1993). In addition, antisense oligonucleotides are considered to be ideal agents for inhibiting viral replication, as they can be specifically targeted to viral RNA sequences, and therefore are not likely to affect host-specific gene expression. Antisense oligonucleotides have been used in cell culture systems to inhibit the replication of a number of viruses, including HIV (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146, 1986; Matsukura et al., Proc. Natl. Acad. Sci. USA 84:7706-7710, 1987; Lisziewicz et al., Proc. Natl. Acad. Sci. USA 89:11209-11213, 1992), influenza (Kabanov et al., FEBS Lett. 259:327-330, 1990), herpes simplex (Smith et al., Proc. Natl. Acad. Sci. USA 83:2787-2791, 1986), vesicular stomatitis (Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652, 1987), and hepatitis B viruses (Goodarzi et al., J. Gen. Virol. 71:3021-3025, 1990; Blum et al., Lancet 337:1230, 1991; Wu et al., J. Biol. Chem. 267(18):12436-12439, 1992; Offensperger et al., EMBO J. 12:1257-1262, 1993). The inhibitory effects of antisense oligonucleotides on viral activity is highly specific. For example, Lisziewicz et al. found that two overlapping 28 nucleotide antisense oligonucleotides inhibited HIV replication, but with a 20-fold difference in efficiency (Lisziewicz et al., Proc. Natl. Acad. Sci. USA 89:11209-11213, 1992).
Hepatitis C virus (HCV) is a positive strand RNA virus with a linear genome of about 9,500 bases. Different isolates show considerable nucleotide (nt) sequence diversity, leading to the subdivision of HCV genomes into at least eight genotypes (Simmonds et al., J Gen Virol, 74:2391-2399, 1993). In all genotypes the viral RNA contains a large open reading frame that encodes a polyprotein precursor of 3010-3033 amino acids (Choo et al., Proc Natl Acad Sci USA, 88:2451-2455, 1991). This precursor is cleaved by cellular and viral proteinases to give rise to the core (C), envelope (E1, E2) and non-structural proteins, e.g., NS2-NS5 (Selby et al., J Gen Virol, 74:1103-1113, 1993). The coding sequence of the RNA genome is preceded by a 5' non-coding region (NCR) of 324-341 nucleotides (Han et al., Proc Natl Acad Sci USA, 88:1711-1715, 1991; Bukh et al., Proc Natl Acad Sci USA, 89:4942-4946, 1992) which is highly conserved among all strains of HCV. The NCR forms a stable secondary structure (Brown et al., Nucl Acids Res, 20:5041-5045, 1992), and provides an internal ribosomal entry site (IRES) which is essential for efficient cap-independent viral translation (Wakita and Wands, J Biol Chem, 269:14205-1421, 1994).
HCV is the major causative agent of post-transfusion hepatitis (Kuo et al., Science 244:362-364, 1989). Persistent HCV infection often leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (Kiyosawa et al., Hepatol. 12:671-675, 1990). Interferon alpha is widely used as an antiviral agent for treatment of chronic HCV infection but its effects have often been found to be limited and transient. (Hoofnagle et al., N. Engl. J. Med. 315:1575-1578, 1986; Yoshioka et al., Hepatol. 16:293-299, 1992). Thus, a need exists for an effective agent for treating HCV infection.